This study has shown that acute icv administration of cannabinoids promotes a long-lasting and robust desensitization of supraspinal CB1Rs, which could be mediated by post-receptor events. The analgesic tolerance that is observed after ip or sc administration of these substances usually requires repeated injections and affects both CB1 and CB2 receptors at spinal and supraspinal levels [10, 61]. This long-term cannabinoid administration produces CB1R desensitization and down-regulation . Specifically, supraspinal CB1R expression diminishes, as does the ability of systemic cannabinoids to induce hypoactivity, hypothermia, and antinociception [5, 8, 9]. Up to 2 weeks are required to recover the initial levels of CB1Rs in the synaptosomal membrane as well as the analgesic response to cannabinoids [5, 6, 9, 62]. Therefore, the analgesic tolerance that follows the repeated systemic administration of cannabinoids can be explained in terms of the loss of surface CB receptors.
Brain CB1Rs, however, desensitize in response to acute doses of agonists; this cannot be explained merely in terms of a permanent loss of receptors. The effect of a single icv-injection of ACEA or WIN55,212-2 on surface CB1Rs is certainly brief. During the analgesic time-course of these agonists, the CB1Rs decreased in the PAG membrane by 60–70%. Most of the internalized CB1Rs bind to GASP1 and are then degraded in the lysosomal compartment [8, 9]. After the analgesic effects of single doses of the cannabinoids cease, the CB1Rs are gradually restored to the surface, probably by both the recycling of a portion of the internalized receptors and the insertion of newly synthesized receptors. As a result, 24 h or 48 h later, the presence of CB1Rs in the membrane is comparable to that seen before the agonist challenge. During this time, the restored CB1Rs become coupled to G proteins, but the analgesic response takes a significantly longer time to be restored: about 14 days. Most relevant, this tolerance is also promoted by cannabinoids such as methanandamide, which cause almost no loss of surface CB1Rs. Thus, it is likely that the analgesic desensitization promoted after several days of systemic treatment with cannabinoids primarily affects receptors at the spinal and peripheral levels, and the associated downregulation of the supraspinal CB1Rs, about 30–50% [5, 62], may be secondary to these effects.
There is compelling evidence that the CB1R couples to and regulates both PTX-sensitive Gi/o proteins and PTX-insensitive Gq/11 and Gz proteins. Thus, the endocannabinoid, 2-arachidonoylglycerol, protects neurons by limiting cyclooxygenase-2 expression, an effect mediated by PTX-sensitive G proteins . WIN55,212-2 shows a more complex pattern of receptor activation. Whereas this agonist affects acetylcholine release in the hippocampus through a PTX-sensitive mechanism , in cultured hippocampal neurons it promotes increases in intracellular calcium via CB1Rs and the PTX-insensitive Gq protein. Interestingly, the latter effect is not reproduced by other cannabinoids, such as THC, CP55,940, 2-arachidonoylglycerol, or methanandamide . These results indicate that after binding the CB1R, cannabinoids may determine the class(es) of G proteins to be activated. Indeed, in a cell line derived from human trabecular meshwork, which is an ocular tissue, WIN55,212-2 was shown to increase intracellular calcium via CB1R and Gq/11 proteins and to increase ERK1/2 phosphorylation via PTX-sensitive Gi/o proteins. In this system, CP55,940 and methanandamide produced the same effects, but they acted via PTX-sensitive Gi/o proteins . Therefore, the CB1R, like the MOR, couples to a series of PTX-sensitive and -insensitive G proteins, and the agonists determine the pattern of G protein activation [56, 58, 64].
Brain CB1Rs mediate the production of analgesia via PTX-sensitive and PTX-insensitive G proteins [ and present study]. The spinal-mediated analgesic action of cannabinoids is mostly mediated via Gi/o proteins. Intrathecal administration of PTX also abolishes the analgesia evoked by icv cannabinoids, indicating that the descending pathways triggered by these substances act at the spinal level through receptors coupled to Gi/o proteins . Signaling via the neural-specific PTX-insensitive Gz protein appears to occur more at the supraspinal level [55, 66]. In fact, supraspinal analgesia mediated by MORs has an important Gz component ; at the spinal level, in contrast, PTX abolishes most MOR-mediated analgesia [66, 67]. Consistent with this observation, the levels of expression of specific regulators of activated Gαz subunits, GAPs, RGSZ1, and RGSZ2, are lower in the spinal cord than in the midbrain [47, 54].
Activation of Gz proteins mediates long-lasting analgesic desensitization of supraspinal CB1Rs. Cannabinoid agonists, such as methanandamide, which apparently do not activate Gq/11 proteins [16, 17], produced desensitization of CB1Rs via activation of Gz proteins. Therefore, it seems that agonists that trigger activation of Gi/o proteins via CB1Rs also activate the PTX-insensitive Gz protein. The unique biochemical and regulatory properties of Gαz subunits account for their strong ability to desensitize GPCR signaling events. The Gz transducer protein, like Gi/o proteins, regulates adenylyl cyclase activity and the gating of certain K+ channels. Gαz, however, is predominantly confined to neuronal cells. The rate of Gαz-GTP hydrolysis is as much as 200-fold slower than that of Gαs-GTP and Gαi-GTP. Therefore, Gz may be resistant to inactivation after receptor activation unless external factors accelerate the rate of Gαz-GTP hydrolysis, much the same way that the GAPs do for many Ras-like proteins. Therefore, inadequate control of Gαz signaling may easily lead to over-regulation of target effectors and subsequent desensitization . Thus, deactivation requires the assistance of specific GAPs to augment the rate of hydrolysis and thus release effector(s) from continuous regulation.
The RGS-Rz subfamily bears the primary responsibility for regulating Gz, and the C-terminus of the MOR associates with a signaling module consisting of HINT1-RGSZ, which helps deactivate MOR-activated Gαz-GTP subunits . This study has shown that brain CB1Rs regulate Gz proteins and associate with the HINT1-RGSZ signaling module, which is involved in the zinc-mediated recruitment of PKCγ . Indeed, PKC has been implicated in the desensitization of CB1Rs by phosphorylation of a serine residue (S317) in the third internal loop . We have observed the in vivo recruitment of PKCγ toward the HINT1-RGSZ module at the C-terminus of CB1R during the intervals when the receptor is uncoupled from regulated transduction. However, the inhibition of this kinase did not prevent the development of acute tolerance, suggesting that other post-receptor mechanisms operate in this process. Most relevant, deregulation of this module brings about increased Gz signaling at the corresponding effector(s) and the development of profound analgesic desensitization of brain MORs [47, 50, 54] and CB1Rs (present study). In contrast, depletion of Gz proteins reduces the analgesic desensitization produced by icv injection of various doses of morphine  and also abolishes acute desensitization of brain CB1Rs and cross-tolerance with morphine. A single icv injection of morphine produces desensitization that lasts for approximately 3 days; however, the cannabinoid agonists studied here desensitized CB1Rs for more than 14 days. Because both of these effects were mediated by the activation of Gz proteins, this observation indicates that CB1R-activated Gz proteins are controlled less efficiently than those activated by the MOR. In agreement with this idea, disruption of the HINT1-RGSZ module led acutely administered morphine to promote a profound and long-lasting desensitization of brain MORs, and most relevant, impaired the analgesic activity of CB1R agonists. Therefore, disruption of Gz regulation brings about a bidirectional supraspinal cross-tolerance between acute doses of morphine and cannabinoids, similar to that attained through chronic and systemic administration of the respective MOR or CB1R agonists [23, 24].
Morphine poorly internalizes MORs and promotes strong analgesic desensitization by stimulating permanent transfer of a part of the receptor-regulated G proteins toward RGS proteins belonging to the R7 and Rz subfamilies [25, 47, 48, 50]. In contrast, DAMGO produces a robust internalization and recycling of MORs, a transient transfer of G proteins toward the RGS proteins, and a low level of analgesic tolerance . Because icv-injected cannabinoids facilitated a reversible transfer of Gα subunits toward RGSZ2 proteins and because the membrane levels of CB1Rs were almost restored within 24 h of their initial challenge, one should expect the resensitization of the analgesic response to these substances, as with DAMGO. However, our results reveal a long-term supraspinal analgesic tolerance even after the CB1R reassociates with these G proteins. This apparent divergence between DAMGO and cannabinoids in the production of tolerance can be explained in terms of the classes of G proteins activated by these agonists after binding to their respective supraspinal receptors. Thus, the analgesic effects of WIN55,212-2 are mediated mostly by Gz proteins, whereas those of DAMGO require Gi/o proteins and, to a lesser extent, Gz [51, 56, 58, 64]. In the absence of Gz activation, the cannabinoids behave as DAMGO, promoting low levels of analgesic tolerance. Therefore, the desensitizing capacity of Gz proteins on post-receptor events predominates over the resensitization caused by reinsertion and G protein-coupling of the internalized CB1Rs in the neural membrane. Thus, it is the coincidence of WIN55,212-2 and morphine at Gz proteins that accounts for their cross-desensitization, whereas the poor regulation of this Gz by DAMGO explains why WIN55,212-2 fails to impair DAMGO-evoked analgesia.
Thus, an inefficient Gαz-GTP deactivation results in desensitization of brain MORs and CB1Rs, suggesting a post-receptor mechanism that appears to be regulated by Gz proteins. However, at the spinal level CB1Rs primarily regulate Gi/o proteins, and in the absence of Gz proteins, tolerance is primarily achieved by reducing the density of active surface receptors. Indeed, this is seen after repeated systemic treatment with cannabinoids (see Introduction).